Excessive oxidation is thought to cause or contribute to heart failure and cardiac arrhythmias, cancer, premature aging, atherosclerosis, Alzheimer's disease, and sepsis. The multifunctional calcium and calmodulin-dependent protein kinase II (CaMKII) now has been identified as a novel molecular target for activation by oxidation. (See Erickson e.g., accepted for publication in Cell, projected publication date May 2, 2008 [hereinafter Erickson e.g., Cell 2008]).
Oxidation activates CaMKII (Erickson e.g., Cell 2008). Activated CaMKII can cause heart failure and arrhythmias and is implicated in progression of cancer, neurological disease, and sepsis. In particular, CaMKII oxidation has been shown to play a pivotal role in cardiac cell death during angiotensin II mediated apoptosis. CaMKII oxidation increases during myocardial ischemia and infarction (Erickson Cell 2008) and causes cellular damage (Yang AJP 2006) and cardiac dysfunction after myocardial infarction (Zhang Nat Med 2005). Additionally, regulation of CaMKII activity by oxidation may play a role in cancer, aging, sepsis, cell development and differentiation, because each of these conditions is marked by increased oxidation and CaMKII activity.
Although oxidation is a bona fide pathological signal in important human diseases, diagnostic measures of oxidative stress suitable for clinical applications are lacking. Another problem in utilizing alternative measures of oxidant stress is that they are not directly linked to disease progression. (See, e.g., Roberts Free Radical Biology and Medicine 2007, (discussing the molecular biology of isoprostanes).) Here, it is shown that oxidation of CaMKII causes enhanced CaMKII activity by preventing refolding of the enzyme into an inactive (resting) conformation. CaMKII is unusual because it is a target for oxidation and CaMKII's role in disease and the mechanism of activation by oxidation are understood. (See Erickson e.g., Cell 2008). Thus, measuring oxidized CaMKII may provide the first opportunity to track oxidation of a biologically active molecule present in peripheral blood or biopsy specimens, in contrast, other measures of oxidative stress are ‘bystander’ molecules not directly implicated in disease pathogenesis (e.g., isoprostanes). In addition to diagnostic clinical applications, researchers in the areas of nerve, muscle, heart, infectious disease and cancer biology may benefit from a reagent that can be used to measure oxidant burden, and in particular oxidant-activated CaMKII.
Although CaMKII is a validated target for heart failure and arrhythmias and is implicated as a causal agent in cancer, neurological diseases and aging, there are currently no available methodologies to non-invasively measure oxidized CaMKII activity. A method to detect oxidized CaMKII may be valuable as a diagnostic tool (e.g., to monitor the success of antioxidant therapy, antibacterial therapy, chemotherapy or future therapies that involve inhibiting CaMKII). In addition, the ability to measure oxidized CaMKII may be invaluable to researchers attempting to connect oxidative stress with CaMKII activation in these fields. Current antibodies only detect either total CaMKII or the phosphorylated form of CaMKII.
Here, an immune serum (rabbit) against the oxidized form of CaMKII was developed and purified by binding to protein A beads. This serum can be used to detect the presence of oxidized CaMKII in heart, blood and other tissues or body fluids using a number of techniques, including Western blot and immunofluorescent imaging. This antiserum may be used to measure oxidized CaMKII in patients from peripheral blood and/or biopsy specimens. In particular, the serum was utilized to detect elevated levels of oxidized CaMKII in blood from mice that serve as models for various human diseases or conditions.